1. Field of the Invention
The present invention relates to purified nucleic acids encoding Antarctic bacteria (Psychrobacter sp.) derived enzymes such as xylanases, which can be a protein, and to purified polypeptides that have high activity and belong to the Family GH10 xylanase-like enzymes. The present invention also provides a genetically recombinant xylanase, with high xylanase activity and having cold adapted activity. The present invention also relates to a process for the production of the recombinant xylanase. The invention also relates to nucleic acids, the corresponding amino acid sequences, constructs, expression vectors or integration vectors containing the DNA molecule, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides. Furthermore, the present invention relates to a method of preparing the cold adapted xylanase by use of recombinant DNA techniques.
2. Description of the Prior Art
Xylans are heteropolysaccharides which form the major part of the hemicellulose present in the plant biomass. Xylan is a polymer of D-xylose linked by β-1,4-xylosidic bonds. Many different side groups could bind to these residues like acetyl, arabinosyl and glucuronosyl residues. Xylan can be degraded to xylose and xylo-oligomers by acid or enzymatic hydrolysis. Enzymatic hydrolysis of xylan produces free sugars without the by-products formed with acid (e.g. furans).
Endoxylanases hydrolyze specifically the backbone of the hemicellulose. In some cases, the side groups may mask the main chain by steric hindrance. Different xylanase activities already described are characterized by their specificity towards their substrate and the length of the oligomers produced.
Enzymes capable of degrading xylan and other plant cell wall polysaccharides are important in various industrial areas. Xylanases are used in the pulp, paper, feed and bakery industries. Other applications include the juice and beer industries, where their ability to catalyse the degradation of the backbone or sidechains of the plant cell wall polysaccharide is utilized. Xylanases could also be used in the wheat separation process. The observed technological effects are, among others, improved bleachability of the pulp, decreased viscosity of the feed or changes in dough characteristics. Others applications for xylanases are enzymatic breakdown of agricultural wastes for production of alcohol fuels, enzymatic treatment of animal feeds for hydrolysis of pentosans and manufacturing of dissolving pulps yielding cellulose.
Xylanases, e.g., endo-β-1,4-xylanase (EC 3.2.1.8), which hydrolyze the xylan backbone chain, have been studied for their use in bleaching lignocellulosic material. For example, in U.S. Pat. No. 5,179,021, the combination of xylanase and oxygen treatment in the bleaching of pulp is disclosed as being particularly useful. WO 92/17573 discloses a substantially pure xylanase derived from Humicola insolens and recombinant DNA encoding said xylanase for use as a baking agent, a feed additive, and in the preparation of paper and pulp. In PCT Application Publication No. WO 92/03541, a method of dissolving hemicellulose with hemicellulases derived from the fungus Trichoderma reesei is disclosed.
There is great interest in discovering different xylanases that will function at the various reaction conditions used in industry for different applications. Much of the research has been directed at discovering thermoactive enzymes. By elevating the temperature, the rates of the reaction are increased. The higher reaction temperatures are appealing for reasons such as sterilization, enhanced reaction rate, and increased availability of substrate, but there is decreased process-energy efficiency. There has been much less work on cold-active xylanases. However, this class of enzymes has received increasing interest because some industrial processes require the use of lower temperatures to avoid altering or denaturing the product. There can also be considerable energy savings for those reactions that can be efficiently conducted at lower temperatures (Kulkurni N, Shendye A, Rao M. 1999. Molecular and biotechnological aspects of xylanases, FEMS Microbiology Review 23: 411-456).
It is an object of the present invention to provide new polypeptides having xylanase activity and nucleic acids encoding the polypeptides.